The ultimate goal of contemporary restorative dentistry is to partially or completely replace compromised or lost tooth tissue using a minimally invasive treatment strategy. In both cases, a complex biomechanical entity is formed at the interface between biomaterials and mineralized hard tissues, which ultimately controls the premature failure of tooth restorations and dental implants.The overall aim of this study was to characterize the micro-pathogenesis of interface failure with mineralized hard tissues, by performing a comprehensive characterization of interaction mechanisms and optimization strategies for clinically relevant and challenging interfaces. Moreover, we have also developed and applied advanced characterization techniques based on recently developed ion-beam sample preparation techniques and quantitative 3D electron microscopy analysis of hard tissue-biomaterial interfaces.The first part of this thesis focused on tooth-glass ionomer interfaces, which, despite their excellent bonding retention in clinical trials, are a challenge to mechanical testing and structural characterization due to their inherent brittleness, reactivity and solubility. Our aim was thus to develop and test new restorative strategies with glass ionomers that were free from their current limitations (papers 2 &amp; 3), based on a better understanding of the chemical interaction at these interfaces (paper 1).We have found through high-resolution microscopy and chemical surface analysis that the self-adhesiveness of RMGIs should be attributed to ionic bonding to hydroxyapatite that remained attached to the partially exposed collagen fibrils, as well as to micro-mechanical interlocking for those RMGIs that additionally hybridize dentin. Moreover, a distinct polycarboxylate gel resulted from an ion-exchange process between the RMGI and dentin as a particular morphologic interfacial feature, the gel-phase. Subsequently, we have determined that the self-etch technique showed very promising in vitro results when applied to glass ionomer-tooth interfaces, since it further enhanced the user-friendliness of RMGIs and lowered their technique-sensitivity, while maintaining desirable characteristics of the conventional etch-and-rinse approach with polyalkenoic acids. Finally, the investigated nano-filled RMGI exhibited adequate bond strength to dentin and enamel, at the same magnitude as other glass ionomers, on the condition that the surface was beforehand treated with the proprietary primer, thus confirming that the non-rinsing approach and specific delivery device favored the ease of use and might reduce the technique sensitivity.The second part of this thesis focused on overcoming the many limitations experienced in our previous studies with the glass ionomer-tooth interface, such as the difficult and labor-intensive sample preparation for conventional high-resolution analysis and the possible interpretation artifacts in such analysis due to reduced sampling, operator bias and/or 2D projection of 3D features. It was also clear that such problems were equally present in other promising, but challenging hard tissues-biomaterial interfaces, which have been studied by separate groups up to the moment, but could benefit from a more unified approach. Consequently, we have chosen to customize and apply newly-developed techniques on such interfaces. We concentrated in particular on sample preparation for high-resolution analysis (paper 4) and exploration of intensively discussed topics in the literature using multi-scale 3D analysis, such as nanoleakage in tooth-biomaterial interfaces (paper 5) and histomorphometric analysis of bone-implant interfaces (paper 6).The FIB/BIB techniques demonstrated to be suitable preparation methodologies of toothbiomaterial interfaces for TEM and nicely complemented information obtained from ultramicrotomy-based techniques. They excelled particularly for more advanced TEM analysis, but not without compromises. Fortunately, most of them could be controlled through judicious preparation and analysis. Subsequently, we have shown that conventional nanoleakage evaluation was heavily influenced by direction, position, and inclination of field-of-view. We have also demonstrated two novel techniques to evaluate nanoleakage quantitatively in 3D and at high resolution, and in a way that was not affected by possible artifacts with conventional techniques. Finally, we have clearly demonstrated that qualitative and quantitative 2D-analysis of bone-implant interfaces may contain severe under- or over-estimation of bone trabeculae, both at meso- and micro-scale. As the heterogeneity of bone-distribution parameters cannot be inferred from single histologic slices of the interface, future studies should either perform a controlled confirmation of their findings using 3D-based techniques, or at least analyze multiple slices produced from the same specimens.